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. 2020 Aug 12;10(1):13662.
doi: 10.1038/s41598-020-70694-8.

Evaluation of cytotoxic T lymphocyte-mediated anticancer response against tumor interstitium-simulating physical barriers

Shu-Ching Chen #  1 , Po-Cheng Wu #  2 , Chiao-Yi Wang  2 , Po-Ling Kuo  3   4   5
Affiliations

Evaluation of cytotoxic T lymphocyte-mediated anticancer response against tumor interstitium-simulating physical barriers (V体育平台登录)

Shu-Ching Chen et al. Sci Rep. .

Abstract

Tumor antigen-specific cytotoxic T lymphocyte (CTL) is a promising agent for cancer therapy. Most solid tumors are characterized by increased interstitial fluid pressure (IFP) and dense collagen capsule, which form physical barriers to impede cancer treatment. However, it remains unclear how CTL-mediated anticancer response is affected at the presence of these obstacles. Using a microfluidic-based platform mimicking these obstacles, we investigated the migration characteristics and performance of anticancer response of CTLs targeting hepatic cancer cells via antigen-specific and allogeneic recognition. The device consisted of slit channels mimicking the narrow interstitial paths constrained by the fibrous capsule and increased IFP was simulated by applying hydrostatic pressure to the tumor center. We found that antigen-specificity of CTLs against the targeted cancer cells determined the cytotoxic efficacy of the CTLs but did not significantly affect the success rate in CTLs that attempted to infiltrate into the tumor center. When increased IFP was present in the tumor center, CTL recruitment to tumor peripheries was promoted but success of infiltration was hindered VSports手机版. Our results highlight the importance of incorporating the physical characteristics of tumor interstitum into the development of CTL-based cancer immunotherapy. .

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VSports在线直播 - Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic and photographic representation of the microfluidic platform simulating physical obstacles in tumor interstitium, which consisted of three 50 μm high main channels interconnected by 5 μm high slit channels. Fresh medium was supplied via the top channel at a flow rate of 0.1 μL/min. CTLs and tumor cells were cultured in the middle and bottom channel respectively, which were referred to as T-cell and cancer-cell channel for clarity. Elevated IFP was simulated by rising the medium-containing syringes that were connected with the cancer-cell channel above the platform. Scale bar: 100 μm.
Figure 2
Figure 2
Tumor-interstitium infiltration and killing of a BNL cell executed by a 2C CD8+ T cell. The T cell and targeted BNL cell were denoted by a yellow and red arrow, respectively. (a) The T cell moved to the opening of slit channel. (b) The T-cell successfully transmigrated through the slit channel and moved to cancer-cell channel. (c) The T cell attached to the targeted BNL cell, and (d) apoptosis of the targeted cell occurred roughly two hours thereafter. The scale bars are 100 μm, and time is expressed as hh:mm:ss.
Figure 3
Figure 3
T-cell recruitment enhanced by apoptosis of cancer cells. Phase contrast images of cultured T cells and HEPA1-6.GP33+ cancer cells acquired (a) before and (b) after apoptosis of the cancer cells located near and in between the slit channel #5 and #6. The cell cluster was marked by the yellow box. Inserts: magnified images of the cell cluster with the apoptotic cells close to the slit channel highlighted by red arrows. Green arrows denote other apoptotic cells in the cluster. Scale bars: 100 μm. Time is expressed as hh:mm:ss. (c) The temporal profile of the cumulative count of the incident when a CTL sought to transmigrate through a slit channel to the cancer-cell channel. The dashed line represents the linear regression of the cumulative counts against time. The local slope of the profile is interpreted as the trend of the CTLs managing to transmigrate at the moment, and the slope of the linear regression is considered as the rate of transmigration owing to background migration. The vertical dash-line denoted when apoptosis of cancer cells denoted by the red arrow in (b) occurred. (d) Histogram of the incidents of transmigration attempt that occurred at individual slit channels throughout the experiment. Channel numbers are defined in (a) and (b).
Figure 4
Figure 4
Distribution of the successful transmigration time of individual CTLs. (a) Scatter plots of the time spent by individual CTLs to transmigrate through the slit channels when the T-cell and cancer-cell channels were cultured with P14 and HEPA1-6.GP33-, P14 and HEAP1-6.GP33+, and 2C and BNL cells, respectively. Data were collected from 2, 2, and 3 individual experiments for the GP33+, GP33−, and BNL cancer cells, respectively. (b) Histogram of the transmigration time when data of the same condition were pooled together. * and *** indicate p < 0.05 and p < 0.001 respectively.
Figure 5
Figure 5
Number of HEPA1-6.GP33+ cells killed by individual P14 CTLs and that of BNL cells killed by individual 2C CTLs.
Figure 6
Figure 6
Simulated hydrodynamics profile in the tumor-interstitium-mimicking device. (a) Computed pressure distribution inside the device when a HP of 20 cmH2O (~ 1960 Pa) was imposed to the cancer-cell channel and a continuous flow rate of 0.1 μL/min was set in the vessel channel. The pressure distributions inside the T-cell and cancer-cell channels were generally homogeneous and an abrupt pressure drop was seen in the slit channels, which were magnified in the inset for clarity. (b) Schematic of T-cell transmigration at the peripheries of the simulated tumor center and the spatial profiles of flow velocity (blue) and HP (green) along a slit channel. The HP gradient inside the channel propelled a continuous flow through the channel at a rate about 14 mm/s, which dropped abruptly at the opening of the slit channel toward the T-cell channel. Note that the slit channel was 30 µm long and only the profile data along one third of the channel length were plotted for clarity.
Figure 7
Figure 7
Morphological characteristics of CTLs close to slit channels when the cancer-cell channel was pressurized. (a) CTLs formed a crowd near the opening of slit channels when various HPs were applied to the cancer-cell channel. (b) The “lollipop”-like appearance of CTLs after the cells failing to transmigrate and leaving the opening of a slit channel.
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